Zone of Inhibition

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Zone of Inhibition
Area around antibiotic disk with no bacterial
growth.
Bacteriocidal no bacteria living in ZOI.
Bacteriostatic bacteria are not reproducing
in ZOI.
Note the lawn of bacteria.
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Bacteria and Antibiotics
Bacteria single-celled organisms that are
prokaryotic (no true nucleus)
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Bacteria and Antibiotics
Bacteria are often used in Biotech to study
genes. Why use bacteria?
Simple to grow
Grow rapidly
24 hours
370C
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Bacteria and Antibiotics
To grow bacteria, need

1. Correct nutrients LB agar (solid) or LB broth
   (liquid).

2. Correct temperature 370C.
3. Can divide every 20 minutes.
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1. Takes time to adjust.

2. 2n
3. Run out of nutrients
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Bacteria and Antibiotics
Antibiotics any chemical which prevent bacteria
from growing.

1. Bacteriocidal

- kills bacteria.

• 2. Bacteriostatic

- prevents bacteria from reproducing. Originals
are not dead!
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Sterile Technique
Preventing contamination - of you by
experiment. - of experiment by you.
Ex. Gloves, keeping lids closed, alcohol, flame.
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Transformation
Changing an organisms traits by adding a new
gene.
Why bacteria? Simple organisms with few genes.
One change in genotype will probably lead to
change in phenotype.
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Ways to get DNA into a cellElectroporation
Using electricity to poke holes in cell membrane
DNA rushes into cell.
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Ways to get DNA into a cellVirus Infection

• Remove disease causing genes from virus.
• Insert gene of interest.
• Virus infects cell with new gene.

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Ways to get DNA into a cellGene Gun

• DNA attached to gold pellet.
• Pellet fired into cell.
• Typically used with plants. Why?

Gene Gun Animation
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Ways to get DNA into a cellHeat Shock
Using heat to open pores in cell membrane. (see
sketch on board).
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Transformation Efficiency
of bacterial cells on plate bacterial cells
possible on plate For heat shock, this number
is lt0.01
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How do you know which cells have been transformed?
Selectable marker gene which gives an obvious
phenotype to transformed cell and allows only
transformed cells to survive. Ex. Antibiotic
resistance genes
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What DNA will we transform into cells?
pVIB luciferase gene encoding
bioluminescence ampr ampicillin-resistance
gene beta-galactasidase which breaks down cell
wall Any bacteria with this gene can grow in
presence of ampicillin (antibiotic)
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What DNA will we transform into cells?
pGREEN GFP Green Fluorescent Protein Also
ampr
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What DNA will we transform into cells?
pUWL500 luciferase and ampr
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What DNA will we transform into cells?
pGEM3z- Just ampr
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What type of bacterial cells are we going to
transform?
E. Coli that are competent. Competent
ready to be transformed.
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How are we going to make competent cells?
Treat cells with CaCl2 In solution CaCl2 ? Ca2
2 Cl- Positively charged Ca2 coats
negatively charged pores. DNA can move into cell.
Heat Shock Animation